Cysteine Proteases as Therapeutic Targets for Neurodegenerative Diseases

The calpain-cathepsin hypothesis (CCH) predicates elevation of calpain-1 (CAPN1) and cathepsin-B (CTSB) as an underlying mechanism in the pathogenesis of Alzheimer’s disease (AD) and related dementia, traumatic brain injury (TBI), and ischemic stroke. The hypothesis is supported by studies with small molecule inhibitors, such as NYC-438, that reduce cognitive deficits in AD mouse models. Though they display efficacy, NYC-438, a nonselective CAPN1/CTSB inhibitor and selective CAPN1 inhibitors reported in the literature exhibit poor brain bioavailability. We hypothesized that the CCH could account for dysfunction of the blood-brain barrier (BBB) and, in particular, brain endothelial cell (BEC) dysfunction. To test this theory and further characterize selective vs nonselective targeting of CAPN1 vs CTSB, we developed selective small molecule inhibitors, and characterized their neuroprotective efficacy in in vitro ischemia-reperfusion injury and neuroinflammatory attenuation in an in vivo mTBI mouse model of oxidative-stress (OS). Various inhibition strategies provided the expected dose-dependent neuroprotection in primary neurons and mitigated the post-mTBI neuroinflammatory surge seen in the OS-mice. Furthermore, we revealed that our in vivo model presents with significant BBB-dysfunction, evidenced by increased sodium fluorescein extravasation and MMP-9 as well as loss in tight junction protein and eNOS. These effects were mitigated by a single injection of our inhibitors 24 hours post trauma. We then isolated BECs from WT and OS mice and saw enhanced susceptibility in the OS-BECs after ischemia-reperfusion injury, suggesting a role for oxidative stress and lipid peroxidation in exacerbating CAPN1/CTSB mediated BBB damage. BECs from WT and OS mice provide a platform to assess the role of CCH in cell viability and tight junction proteins, and provides support for targeting CAPN1/CTSB in protecting the BBB, either in early life trauma, such as TBI, or in ADRD itself. This work establishes CCH inhibition as a potential prophylactic therapeutic strategy to protect the neurovascular unit

Interaction of oxidative stress and neurotrauma in ALDH2−/− mice causes significant and persistent behavioral and pro-inflammatory effects in a tractable model of mild traumatic brain injury

Oxidative stress induced by lipid peroxidation products (LPP) accompanies aging and has been hypothesized to exacerbate the secondary cascade in traumatic brain injury (TBI). Increased oxidative stress is a contributor to loss of neural reserve that defines the ability to maintain healthy cognitive function despite the accumulation of neuropathology. ALDH2-/- mice are unable to clear aldehyde LPP by mitochondrial aldehyde dehydrogenase-2 (Aldh2) detoxification and provide a model to study mild TBI (mTBI), therapeutic interventions, and underlying mechanisms. The ALDH2-/- mouse model presents with elevated LPP-mediated protein modification, lowered levels of PSD-95, PGC1-α, and SOD-1, and mild cognitive deficits from 4 months of age. LPP scavengers are neuroprotective in vitro and in ALDH2-/- mice restore cognitive performance. A single-hit, closed skull mTBI failed to elicit significant effects in WT mice; however, ALDH2-/- mice showed a significant inflammatory cytokine surge in the ipsilateral hemisphere 24 h post-mTBI, and increased GFAP cleavage, a biomarker for TBI. Known neuroprotective agents, were able to reverse the effects of mTBI. This new preclinical model of mTBI, incorporating significant perturbations in behavior, inflammation, and clinically relevant biomarkers, allows mechanistic study of the interaction of LPP and neurotrauma in loss of neural reserve